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1.
The prolate icosahedral capsid geometry of wild type bacteriophage T4D has been determined by direct visualization of the triangular faces in stereoimages of transmission electron micrographs of phage particles. Bacteriophage T4 was prepared for transmission electron microscopy (TEM) following a protocol of freeze-fracturing, deep-etching (FDET) and replication by vertical deposition (80 degrees angle) of a thin platinum-carbon (Pt-C) metal layer of 1.01 nm. From direct statistical measurements of the ratio of the head length to width and of stereometric angles on T4 heads, we have estimated a Q number of 21. This confirms previous indirect studies on T4 and agrees with determinations on bacteriophage T2. Many of the structural features of T4 observed in FDET preparations differ significantly from those observed by classical negative staining methods for TEM imaging. Most important among the differences are the conformation of the baseplate (a closed rosebud) and the positioning of the tail fibers (retracted). The retracted position of the tail fibers in the FDET preparations has been confirmed by negatively staining phage previously fixed suspended in solution with 2% glutaraldehyde. The FDET protocols appear to reveal important structural features not seen in negative stained preparations. These have implications for bacteriophage T4 conformation in solution, viral assembly and phage conformation states prior to tail contraction and DNA ejection.  相似文献   

2.
The wac gene product (gpwac) or fibritin of bacteriophage T4 forms the six fibers that radiate from the phage neck. During phage morphogenesis these whiskers bind the long tail fibers (LTFs) and facilitate their attachment to the phage baseplate. After the cell lysis, the gpwac fibers function as part of an environmental sensing device that retains the LTFs in a retracted configuration and thus prevents phage adsorption in unfavorable conditions. A comparative analysis of the sequences of 5 wac gene orthologs from various T4-type phages reveals that the approximately 50-amino-acid N-terminal domain is the only highly conserved segment of the protein. This sequence conservation is probably a direct consequence of the domain's strong and specific interactions with the neck proteins. The sequence of the central fibrous region of gpwac is highly plastic, with only the heptad periodicity of the coiled-coil structure being conserved. In the various gpwac sequences, the small C-terminal domain essential for initiation of the folding of T4 gpwac is replaced by unrelated sequences of unknown origin. When a distant T4-type phage has a novel C-terminal gpwac sequence, the phage's gp36 sequence that is located at the knee joint of the LTF invariably has a novel domain in its C terminus as well. The covariance of these two sequences is compatible with genetic data suggesting that the C termini of gpwac and gp36 engage in a protein-protein interaction that controls phage infectivity. These results add to the limited evidence for domain swapping in the evolution of phage structural proteins.  相似文献   

3.
Concentrated suspensions of T4 phage crystallize spontaneously in the absence of tryptophan. The crystals, in one plane, contain repeated bilayers of phage in head to head and tail to tail contact, probably with tail fibers retracted. In the plane of each layer there is hexagonal packing of the phage.  相似文献   

4.
At 346 kbp in size, the genome of a jumbo bacteriophage vB_KleM-RaK2 (RaK2) is the largest Klebsiella infecting myovirus genome sequenced to date. In total, 272 out of 534 RaK2 ORFs lack detectable database homologues. Based on the similarity to biologically defined proteins and/or MS/MS analysis, 117 of RaK2 ORFs were given a functional annotation, including 28 RaK2 ORFs coding for structural proteins that have no reliable homologues to annotated structural proteins in other organisms. The electron micrographs revealed elaborate spike-like structures on the tail fibers of Rak2, suggesting that this phage is an atypical myovirus. While head and tail proteins of RaK2 are mostly myoviridae-related, the bioinformatics analysis indicate that tail fibers/spikes of this phage are formed from podovirus-like peptides predominantly. Overall, these results provide evidence that bacteriophage RaK2 differs profoundly from previously studied viruses of the Myoviridae family.  相似文献   

5.
Three types of reagents were used to determine the structural role and location of the polyglutamate portion of the Escherichia coli T4D bacteriophage baseplate dihydropteroyl hexaglutamate. These reagents were examined for their effect in vitro on some of the final steps in phage baseplate morphogenesis. The reagents were (i) a series of oligopeptides composed solely of glutamic acid residues but with various chemical linkages and chain lengths; (ii) a homogeneous preparation of carboxypeptidase G1, an exopeptidase that hydrolyzes carboxyl-terminal glutamates (or aspartates) from simple oligopeptides, including the gamma-glutamyl bonds on folyl polyglutamates as well as the bond between the carboxyl group of the p-aminobenzoyl moiety and the amino group of the first glutamic acid residue of folic acid; and (iii) antisera prepared against a polyglutamate hapten. All three types of reagent markedly inhibited the attachment of the phage long tail fibers to the baseplate. Other steps in baseplate assembly such as the addition of T4D gene 11 or gene 12 products were not affected by any of these reagents. These results indicate that the polyglutamate portion of the folate is located near the attachment site on the bacteriophage baseplate for the long tail fibers.  相似文献   

6.
The protein component of the T-even bacteriophage coat which binds the phage-specific dihydropteroyl polyglutamate has been identified as the phage-induced dihydrofolate reductase. Dihydrofolate reductase activity has been found in highly purified preparations of T-even phage ghosts and phage substructures after partial denaturation. The highest specific enzymatic activity was found in purified tail plate preparations, and it was concluded that this enzyme was a structural component of the phage tail plate. Phage viability was directly correlated with the enzymological properties of the phage tail plate dihydrofolate reductase. All reactions catalyzed by this enzyme which changed the oxidation state of the phage dihydrofolate also inactivated the phage. Properties of two T4D dihydrofolate reductase-negative mutants, wh1 and wh11, have been examined. Various lines of evidence support the view that the product of the wh locus of the phage genome is normally incorporated into the phage tail structure. The effects of various dihydrofolate reductase inhibitors on phage assembly in in vitro complementation experiments with various extracts of conditional lethal T4D mutants have been examined. These inhibitors were found to specifically block complementation when added to extracts which did not contain preformed tail plates. If tail plates were present, inhibitors such as aminopterin, did not affect further phage assembly. This specific inhibition of tail plate formation in vitro confirms the analytical and genetic evidence that this phage-induced "early" enzyme is a component of the phage coat.  相似文献   

7.
By recombination between bacteriophage T4 wh2, a dihydrofolate reductaseless mutant, and T6, I have prepared T4 wh(T6), a T4 strain which codes for the T6-specific soluble dihydrofolate reductase. This strain has the heat sensitivity of T6, not T4, which provides direct evidence that the wh gene codes for both the soluble dihydrofolate reductase and the structural dihydrofolate reductase which is a constituent of T-even phage tail plates.  相似文献   

8.
We have calculated the translational and rotational frictional coefficients of structures related to T2 and T4 bacteriophage, using the theoretical framework developed in the preceding two papers. The structures considered were models for tail-fiberless phage, and for whole phage with fibers in the extended and retracted portions. We also computed and compared with the experiment the changes in translational frictional coefficient produced by successive addition of 1–6 fibers to the fiberless particle. Agreement with experimental results is markedly improved over previous theoretical efforts, especially with respect to the effect of tail-fiber extension. Some significant discrepancies remain, however, in the comparison of fiber-retracted and fiberless phage.  相似文献   

9.
A recently isolated phage, vB_EcoP_SU10 (SU10), with the unusual elongated C3 morphotype, can infect a wide range of Escherichia coli strains. We have sequenced the genome of this phage and characterized it further by mass spectrometry based proteomics, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and ultra-thin section electron microscopy. The genome size is 77,327 base pairs and its genes, and genome architecture, show high similarity to the phiEco32 phage genes and genome. The TEM images reveal that SU10 have a quite long tail for being a Podoviridae phage, and that the tail also changes conformation upon infection. The ultra-thin section electron microscopy images of phages at the stage of replication within the host cell show that the phages form a honeycomb-like structure under packaging of genomes and assembly of mature capsids. This implies a tight link between the replication and cutting of the concatemeric genome, genome packaging, and capsid assembly. We have also performed a phylogenetic analysis of the structural genes common between Podoviridae phages of the C1 and C3 morphotypes. The result shows that the structural genes have coevolved, and that they form two distinct groups linked to their morphotypes. The structural genes of C1 and C3 phages appear to have diverged around 280 million years ago applying a molecular clock calibrated according to the presumed split between the Escherichia – Salmonella genera.  相似文献   

10.
Chemically Induced Cofactor Requirement for Bacteriophage T4D   总被引:1,自引:0,他引:1       下载免费PDF全文
The treatment of bacteriophage T4D with 2-hydroxy-5-nitrobenzyl bromide, a specific reagent for alkylating the indole ring of tryptophan residues, converts these particles from a cofactor-independent form to a cofactor-sensitive form. These treated T4D particles phenotypically resemble T4B particles in certain respects. Their ability to form plaques on minimal medium plates is increased by the addition of l-tryptophan and is inhibited by the addition of indole. In liquid medium, their rate of adsorption is dependent on the presence of the cofactor l-tryptophan. l-Tryptophan-requiring phage have been produced by in vitro assembly of treated tail-fiberless particles of a T4D amber mutant plus untreated tail fiber preparation. When treated tail fibers were used with untreated tail-fiberless particles, the newly assembled particles did not require cofactor. A model of the tail structure of all the T-even bacteriophages is presented which postulates that the active configuration of the tail fibers requires that there be either (i) an endogenous tryptophan residue of the phage particle itself or (ii) an exogenously added l-tryptophan molecule complexed with a specific tryptophan receptor site, most likely on the phage base plate.  相似文献   

11.
The majority of bacteriophages protect their genetic material by packaging the nucleic acid in concentric layers to an almost crystalline concentration inside protein shells (capsid). This highly condensed genome also has to be efficiently injected into the host bacterium in a process named ejection. Most phages use a specialized complex (often a tail) to deliver the genome without disrupting cell integrity. Bacteriophage T7 belongs to the Podoviridae family and has a short, non-contractile tail formed by a tubular structure surrounded by fibers. Here we characterize the kinetics and structure of bacteriophage T7 DNA delivery process. We show that T7 recognizes lipopolysaccharides (LPS) from Escherichia coli rough strains through the fibers. Rough LPS acts as the main phage receptor and drives DNA ejection in vitro. The structural characterization of the phage tail after ejection using cryo-electron microscopy (cryo-EM) and single particle reconstruction methods revealed the major conformational changes needed for DNA delivery at low resolution. Interaction with the receptor causes fiber tilting and opening of the internal tail channel by untwisting the nozzle domain, allowing release of DNA and probably of the internal head proteins.  相似文献   

12.
Assembly of the long tail fibers of the Escherichia coli bacteriophage T4 requires the catalytic action of two auxiliary proteins. It was found that a gene of the entirely unrelated phage lambda codes for a protein which can substitute for one of these T4 polypeptides, protein 38. The lambda gene was designated tfa (tail fiber assembly). Protein 38 consists of 183 residues, and the Tfa protein consists of 194 residues; the two polypeptides are about 40% homologous. Although the tfa gene is dispensable for the growth of phage lambda, these results indicate that it may have a function in lambda morphogenesis.  相似文献   

13.
The tail of bacteriophage T4 undergoes large structural changes upon infection while delivering the phage genome into the host cell. The baseplate is located at the distal end of the contractile tail and plays a central role in transmitting the signal to the tail sheath that the tailfibers have been adsorbed by a host bacterium. This then triggers the sheath contraction. In order to understand the mechanism of assembly and conformational changes of the baseplate upon infection, we have determined the structure of an in vitro assembled baseplate through the three-dimensional reconstruction of cryo-electron microscopy images to a resolution of 3.8 Å from electron micrographs. The atomic structure was fitted to the baseplate structure before and after sheath contraction in order to elucidate the conformational changes that occur after bacteriophage T4 has attached itself to a cell surface. The structure was also used to investigate the protease digestion of the assembly intermediates and the mutation sites of the tail genes, resulting in a number of phenotypes.  相似文献   

14.
对产L-天冬氨酸酶大肠埃希菌噬菌体进行分离和生理特性研究,有助于为生产过程中噬菌体污染的防治提供指导。采用双层平板法对噬菌体进行分离纯化。利用透射电镜观察噬菌体形态。进行噬菌体全基因组测序和比对。通过测定不同处理条件下噬菌体活性,研究温度、pH、有机溶剂氯仿、去垢剂SDS对噬菌体的影响。从噬菌体污染的L 天冬氨酸酶生产菌种大肠埃希菌HY-05C发酵培养液中分离出1株噬菌体。电镜结果表明,该噬菌体由呈多面体对称的头部和极短的尾部构成。基因组测序和比对结果表明,噬菌体与T7样噬菌体的相似性最高。生理特性研究表明,噬菌体对高温和去垢剂SDS敏感,对有机溶剂氯仿不敏感;最适pH为7.0,碱性条件下活力较为稳定,酸性条件容易失活。噬菌体保藏编号为CICC 80001。  相似文献   

15.
The tail of bacteriophage T4 consists of a contractile sheath surrounding a rigid tube and terminating in a multiprotein baseplate, to which the long and short tail fibers of the phage are attached. Upon binding of the fibers to their cell receptors, the baseplate undergoes a large conformational switch, which initiates sheath contraction and culminates in transfer of the phage DNA from the capsid into the host cell through the tail tube. The baseplate has a dome-shaped sixfold-symmetric structure, which is stabilized by a garland of six short tail fibers, running around the periphery of the dome. In the center of the dome, there is a membrane-puncturing device, containing three lysozyme domains, which disrupts the intermembrane peptidoglycan layer during infection.  相似文献   

16.
The characteristics of pure preparations of short-tail fibers of bacteriophage T4 have been studied in the optical and electron microscope. Three main structures were observed: 1) spheres of 8.1 nm diameter; 2) fibers 43 nm long and 3.8 nm thick; and 3) fibers 54 nm long and 3.2 nm thick. Both types of fibers exhibited a regular beaded appearance. The 43-nm fibers were the most abundant structure. During the process of purification of the short-tail fibers, the formation of aggregates was observed each time the material containing the short-tail fibers was dialyzed against saline solutions. These aggregates became increasingly fibrous (as observed in the optical microscope) as the material used was increasingly enriched in short-tail fibers. Finally, most of the aggregates were of the fibrous type when they were formed from a purified preparation of short-tail fibers. In the electron microscope, it was found that the filamentous aggregates were organized in well-defined bundles. The amino acid composition of the highly purified short-tail fibers was also determined. Among the known fibrous proteins, the ones that most resemble the amino acid composition of the short-tail fibers are actin and fibrinogen. These observations are discussed in relation to the T4 short-tail fiber structure and their localization on the hexagonal baseplate of the T4 tail structure.  相似文献   

17.
The collar and whiskers of bacteriophage T4 extend outward from the top of the tail and play a role in regulating retraction of the tail fibers (Conley &; Wood, 1975). The collar and whiskers also are required for efficient tail fiber attachment during phage assembly. The structural gene for the collar/whisker protein is called wac. In vitro, infected-cell extracts that contain tail fibers activate whiskerless (wac) tail fiberless particles and ordinary (wac+) tail fiberless particles at equal rates if the extracts contain the wac+ gene product. However, extracts that contain tail fibers but no wac+ gene product activate wac particles about ten times more slowly. In vivo, whiskers are not essential for plaque formation, but a wac mutation causes a delay in the appearance of intracellular phage and a fivefold decrease in the burst size of infectious particles.The effect of the whiskers on tail fiber attachment is due to an interaction between the whisker and the distal half of the tail fiber, similar if not identical to the interaction that controls tail fiber retraction in complete phage. The following observations support this view: a slow rate of in vitro tail fiber attachment similar to that described above is seen with wac+ particles when they are pretreated with anti-whisker serum, or when the tail fibers carry a mutational alteration in gp36, a structural protein in the distal half fiber near the central kink. Lack of whiskers does not affect the slow rate of attachment of proximal half fibers to the baseplate of fiberless particles, but lack of whiskers greatly decreases the rate at which particles with attached proximal half fibers are activated by addition of distal half fibers. Since whiskers normally are attached to the phage only after head—tail union (Coombs &; Eiserling, 1977; Terzaghi et al., 1978), these findings explain why tail fibers do not attach efficiently to the baseplates of free tails.  相似文献   

18.
The distal part of the long tail fiber of Escherichia coli bacteriophage T4 consists of a dimer of protein 37. Dimerization requires the catalytic action of protein 38, which is encoded by T4 and is not present in the virion. It had previously been shown that gene tfa of the otherwise entirely unrelated phage lambda can functionally replace gene 38. Open reading frame (ORF) 314, which encodes a protein that exhibits homology to a COOH-terminal area of protein 37, is located immediately upstream of tfa. The gene was cloned and expressed in E. coli. An antiserum against the corresponding polypeptide showed that it was present in phage lambda. The serum also reacted with the long tail fibers of phage T4 near their free ends. An area of the gene encoding a COOH-terminal region of ORF 314 was recombined, together with tfa, into the genome of T4, thus replacing gene 38 and a part of gene 37 that codes for a COOH-terminal part of protein 37. Such T4-lambda hybrids, unlike T4, required the presence of outer membrane protein OmpC for infection of E. coli B. An ompC missense mutant of E. coli K-12, which was still sensitive to T4, was resistant to these hybrids. We conclude that the ORF 314 protein represents a subunit of the side tail fibers of phage lambda which probably recognize the OmpC protein. ORF 314 was designated stf (side tail fiber). The results also offer an explanation for the very unusual fact that, despite identical genomic organizations, T4 and T2 produce totally different proteins 38. An ancestor of T4 from the T2 lineage may have picked up tfa and stf from a lambdoid phase, thus possibly demonstrating horizontal gene transfer between unrelated phage species.  相似文献   

19.
W. Boontje  J. Greve  Joh. Blok 《Biopolymers》1978,17(11):2689-2702
Transient electric birefringence measurements of the bacteriophages T2L0 and T6 were performed under such conditions that the tail fibers are extended. The data obtained are compared to previously reported data for T4B. For all T-even phages the degree of extension of the tail fibers is a function of pH, ionic strength, and temperature. For T4B, much higher ionic strengths are needed than for T2L0 and T6 to accomplish complete tail-fiber extension. The rotational diffusion coefficients of the phages with fully extended fibers are equal to 120 ± 3 sec?1, 132 ± 5 sec?1, 157 ± 4 sec?1 for T2L0, T4B, and T6, respectively. The respective optical anistropies are ? (2.66 ± 0.05) × 10?4, and ? (3.07 ± 0.15) × 10?4. The differences in the rotational diffusion coefficient and optical anisotropy arise because the conformation of the fully extended tail fibers is different for the three phages. The tail fibers of T2L0 project further into the solution (away from the head) than do those of T4B and T6. The apparent permanent dipole moments of T2L0 and T6 decrease with increasing ionic strength. This decrease is caused by the screening of the surface charges on the phage body by the counter-ions in the solution. The biological relevance of this decrease is illustrated by the fact that the adsorption rate of T6 phages to E. coli B bacteria shows a similar dependence of ionic strength. Evidence is pressented that the tail fibers may move more or less independently of the phage body when an electric field is applied to the suspension.  相似文献   

20.
L M Kozloff  M Lute    L K Crosby 《Journal of virology》1975,16(6):1391-1400
Two different proteins with high affinities for the pteridine ring of folic acid have been used to determine the location of this portion of the folate molecule in the tail plate of T4D and other T-even bacteriophage particles. The two proteins used were (i) antibody specific for folic acid and (ii) the folate-binding protein from bovine milk. Both proteins were examined for their effect on various intact and incomplete phage particles. Intact T2H was weakly inactivated by the antiserum but not by the milk protein. No other intact T-even phage, including T4D, was affected by these two proteins. When incomplete T4D particles were exposed in an in vitro morphogenesis system, it was found that neither of the two proteins affected either the addition of the long tail fibers to fiberless particles or the addition of tail cores to tail plates. On the other hand, these two proteins specifically blocked the addition of T4D gene 11 product to the bottom of T4D baseplates. After the addition of the gene 11 protein, these two reagents did not inhibit the further addition of the gene 12 protein to the baseplate. It can be concluded that the phage folic acid is a tightly bound baseplate constituent and that the pteridine portion of the folic acid is largely covered by the gene 11 protein.  相似文献   

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